Typically in the area of chromatographic separations and electrophoresis gels, polysaccharide polymers such as agarose, are used to make gel media by thermally phase separating the polymer from an aqueous solution. This can be done because these polymers have a melting point and a gel point that is above room temperature, preferably above 30° C.
To process agarose for example, the polymer must be heated above its melting temperature, which is about 92° C., in the presence of water. At that temperature the polymer melts and the molten polymer is then solvated by water to form a solution. The polymer remains soluble in water as long as the temperature is above the polymer's gel point, which is about 43° C. At and below the gel point, the polymer phase separates and becomes a hydrogel that takes on whatever shape the solution was in just before gelling. Additionally, as the agarose approaches its gel point, the viscosity of the solution becomes higher and higher as the hydrogel begins to form.
Traditionally, for electrophoresis gels, the polysaccharide is poured onto a flat substrate and allowed to cool forming a sheet of polysaccharide material on which the electrophoresis process can occur.
For polysaccharide beads, such as are used in chromatography media, the heated solution is kept above its gel point and it is stirred into an immiscible, heated fluid such as mineral or vegetable oil to form beads. The two-phased material (beads of agarose in the immiscible fluid) is then cooled and the beads are recovered. The beads can then be used as is for size exclusion chromatography or further processed by crosslinking, addition of various capture chemistries such as affinity chemistries or ligands, positive or negative charge, hydrophobicity or the like or combinations of crosslinking and chemistries.
Some have tried to use agarose to form a coating on or in a structure rather than as a solid article itself. For instance, according to Cerro et al., Biotechnol. Prog 2003, 19 921-927 (Use of ceramic monoliths as stationary phase in affinity chromatography), thin, surface active only agarose coatings on ceramic monoliths were created by impregnating the monolith with the traditional hot solution of agarose, followed by removal of excess hot agarose solution from the cells within the monolith using compressed air and subsequently cooling the monolith to gel the agarose coating.
One of the major problems with this coating process is that the coatings are difficult to effect on porous materials. In the article mentioned above, the agarose had to be applied in a heated state (thus requiring a substrate that is heat stable). A further problem is that only thin coatings that have only surface activity can be created. In part this may be due to the method used for removing excess agarose. It may also be a function of the agarose gel point and the higher viscosity that occurs as the temperature of the agarose approaches the gel point. Moreover the prior art process is very difficult if not impossible with substrates having pores that are relatively small in comparison to the cell size of the monoliths of the prior art. The reason for these difficulties is that in some cases, air cannot be readily forced through certain porous materials without disrupting or otherwise damaging the porous structure as is the case with certain fabrics or porous membranes. Therefore relatively porous, rigid monolithic structures must be used.
WO 00/44928 suggests forming a temperature stable agarose solution through the use of high levels (e.g. 8M) of chaotropes such as urea. Agarose of this invention is imbibed into a porous support to form a continuous phase. Water is carefully added such that a thin gel layer forms at the interfaces between the agarose solution and the added water. The gel layer prevents migration of the agarose but allows further migration of the water and urea molecules out of the agarose solution into the added water. This process continues until the agarose solution turns into a gel within the interstices of the pores of the porous substrate.
One major problem with this prior art method is that the process by which it is made causes the pores of the substrate to be substantially blocked, severely limiting convective flow through the porous support.
What is desired is a method for making gels, coatings and beads of polysaccharides, especially agarose that overcome the problems of the prior art. More particularly, what is desired is the ability to create coatings on relatively small pored porous articles (0.1-500 microns pore size) that allows for good convective flow through the porous structure with diffusive flow within the agarose itself. Another desire is to form beads and other shaped agarose articles without the need for maintaining high temperatures while doing so.
By using the method of the present invention relatively thick, porous coatings on surfaces can be achieved easily, including the surface of porous materials that are capable of both convective and diffusive flows. Additionally, beads, gels and the like can be formed at or near room temperature.